Long-term goal: To elucidate the cellular and molecular basis of excess scar formation during wound repair. This application will focus on the role of altered expression of plasminogen activator inhibitor-1 (PAI-1) in collagen over-production by keloidfibroblasts. Proteolytic degradation of the provisional fibrin matrix and remodeling of the newly formed collagen-containing scar tissues are essential features in injury repair. Keloids, resulting from improper wound healing, are the extreme form of skin fibrosis with unknown etiology. Using a 3-dimensional fibrin gel culture system, we discovered that keloid fibroblasts are defective in fibrin degradation due to PAl-1 over-expression. In the previous granting period, we established, both in vitro and in vivo, that PAI-1 overexpression is phenotypic of keloid fibroblasts and is causal in the elevated collagen accumulation. Reducing PAI-1 activity also abolishes the elevated collagen accumulation in keloid fibroblasts (Tuan et al., Am J Pathol 2003). In addition, we demonstrated that, in vivo, PAI-1 increases as fetal mouse skin wounds transition from scarless (El5) to scar-forming (E18 and after) repair, and aprotinin, a uPA/plasmin inhibitor, causes scar formation in E15 fetal skin wounds (Huang et al., WRR 2002). PAI-1 is the major inhibitor of the plasminogen activator (PA)/plasmin system. This system is central to fibrin degradation, cell adhesion and migration, and metalloproteinase (MMP) activation, which is essential in collagen turnover. Thus, we hypothesize that "PAl-1 contributes to elevated collagen accumulation in keloid fibroblasts by inhibiting MMP activation and or by modulating uPA-mediated cell adhesion". PAI-1 is a down stream target of TGF-beta, and keloid fibroblasts exhibit TGF-beta-mediated differences in matrix contraction and collagen synthesis. Thus, we also hypothesize that "an altered TGF-beta signaling pathway and or utilization of PAI-1 promoter response elements are responsible for increased PAI-1 expression in keloidfibroblasts" The established evidence and the unique experimental models will allow us to test these hypotheses through the following specific aims: Aim I: To investigate the role of PAI-1 increase in MMP- and cell adhesion-mediated collagen accumulation in keloid fibroblasts. Aim II: To determine the biological mechanism of PAI-1 increase resulting from altered TGF-beta signaling events and/or difference in PAI-1 promoter utilization in keloid fibroblasts.